Statement of work The majority of diseases that afflict the central nervous system (CNS) are poorly treated or incurable. The blood- brain barrier (BBB) is increasingly recognized as a contributor to CNS disease and represents an intriguing therapeutic target. However, these efforts are hampered by a poor understanding of BBB function. Using BBB endothelial cells derived from induced pluripotent stem cells (iPSCs), we recently conducted gene expression profiling to identify genes that are upregulated in concurrence with a substantial increase in BBB properties. The purpose of this grant is to validate the importance of a subset of these genes for BBB function using biochemical and genome editing techniques. Aim 1 will focus on knocking out each gene from iPSCs, followed by BBB differentiation and functional analyses. Aim 2 will perform co-immunoprecipitation/mass spectrometry experiments to identify proteins that interact with IQGAP2, which is the most highly upregulated gene from the initial screen. Outcomes from this proposal will lead to an improved understanding of essential contributors to the BBB phenotype.